Duplex real-time PCR assay for quantifying mitochondrial DNA deletions in laser microdissected single spiral ganglion cells.

Laser microdissection (LMD) has been used to isolate groups of cells and single cells from numerous tissues. In this chapter, we describe a technique for isolating individual spiral ganglion cells from archival formalin-fixed, celloidin-embedded (FFCE) human temporal bone sections. The DNA isolated from these single cells is suitable for analysis with a duplex real-time polymerase chain reaction (PCR) methodology to quantify the mitochondrial DNA (mtDNA) deletion level present.

Dual immunofluorescence staining of proteoglycans in human temporal bones.

OBJECTIVES/HYPOTHESIS: Immunofluorescence staining methods have been developed to study the distribution of macromolecules in archival formalin-fixed celloidin-embedded human temporal bone tissues. The aim of this study was to investigate the feasibility of utilizing this approach to evaluate the codistribution of more than one molecule of interest in a single tissue section. STUDY DESIGN: Retrospective study of proteoglycan codistribution in archival human temporal bone tissues. METHODS: The chondroitin sulfate and keratan sulfate proteoglycans were selected for evaluating this methodology. Human tissues with known proteoglycan staining patterns were studied as controls. Thirty-one formalin-fixed celloidin-embedded archival human temporal bones were evaluated, and the observations in 11 specimens are described. A dual immunofluorescence staining method was developed using primary antibodies of differing isotypes and secondary antibodies labeled with fluorophores having nonoverlapping emission characteristics. RESULTS: The specificity of the dual immunofluorescence technique for chondroitin sulfate and keratan sulfate proteoglycans was demonstrated in control tissues and confirmed through inhibition studies. The normal human tectorial membrane exhibited intense chondroitin sulfate staining. Cochlear and vestibular hair cells exhibited predominantly keratan sulfate staining. Keratan sulfate staining predominated in spiral ganglion cell bodies and fibers. Alterations in the normal distribution pattern of proteoglycans were observed in cases of presbycusis and otosclerosis. CONCLUSIONS: The dual immunofluorescence staining methodology can be used to study archival formalin-fixed celloidin-embedded human temporal bone tissues. This technique may be applied to the evaluation of other molecules in archival human temporal bone tissues and lead to improvement in our understanding of the function of these molecules and their role in disease processes.

Proteomic analysis of formalin-fixed celloidin-embedded whole cochlear and laser microdissected spiral ganglion tissues.

CONCLUSION: The results of this study demonstrate that proteomic analysis can be successfully performed on formalin-fixed celloidin-embedded (FFCE) archival human cochlear tissues. OBJECTIVE: To investigate the feasibility of analyzing protein expression in archival cochlear tissues. MATERIAL AND METHODS: A new methodology, referred to as Liquid Tissue(TM), was used to extract proteins from human cochlear tissue sections and spiral ganglion tissue isolated by laser microdissection (LMD). Protein identification was performed by bioinformatic analysis of high resolution tandem mass spectrometric data from fractionated tryptic peptide samples. RESULTS: Twenty-six proteins were identified with a minimum of 2 unique peptides and 450 proteins were identified with 1 unique peptide at a confidence level of 95% in cochlear tissue. Ten proteins were identified with a minimum of 2 unique peptides and 485 proteins were identified with 1 unique peptide at a confidence level of 95% in spiral ganglion tissue.

Major arc mitochondrial DNA deletions in cytochrome c oxidase-deficient human cochlear spiral ganglion cells.

CONCLUSIONS: This study suggests that cytochrome c oxidase subunit 3 (COX 3) expression is diminished in spiral ganglion cells from individuals with presbycusis. In addition to the mitochondrial DNA (mtDNA) common deletion (CD), other deletions involving the mtDNA major arc contribute to the observed deficit in COX 3 expression. OBJECTIVES: To assess COX 3 deficiency in spiral ganglion cells from individuals with presbycusis and to determine whether deletions other than the CD contribute to this deficiency. METHODS: COX 3 immunofluorescence staining of archival human temporal bone tissue sections from individuals with presbycusis and from age-matched normal-hearing individuals was performed and the intensity of spiral ganglion cell immunostaining was measured. Single COX 3-deficient spiral ganglion cells were isolated by laser microdissection (LMD) and the DNA was analyzed with duplex real-time PCR assays to assess the CD level and the total mtDNA major arc deletion level. RESULTS: A statistically significant difference (p = 0.021) in the mean intensity of COX 3 immunofluorescence staining of spiral ganglion cells was observed between individuals with presbycusis and normal-hearing controls. The total mtDNA major arc deletion level was greater than the CD level in COX 3-deficient spiral ganglion cells.

Pathogenic natural antibodies recognizing annexin IV are required to develop intestinal ischemia-reperfusion injury.

Intestinal ischemia-reperfusion (IR) injury is initiated when natural IgM Abs recognize neo-epitopes that are revealed on ischemic cells. The target molecules and mechanisms whereby these neo-epitopes become accessible to recognition are not well understood. Proposing that isolated intestinal epithelial cells (IEC) may carry IR-related neo-epitopes, we used in vitro IEC binding assays to screen hybridomas created from B cells of unmanipulated wild-type C57BL/6 mice. We identified a novel IgM mAb (mAb B4) that reacted with the surface of IEC by flow cytometric analysis and was alone capable of causing complement activation, neutrophil recruitment and intestinal injury in otherwise IR-resistant Rag1(-/-) mice. mAb B4 was found to specifically recognize mouse annexin IV. Preinjection of recombinant annexin IV blocked IR injury in wild-type C57BL/6 mice, demonstrating the requirement for recognition of this protein to develop IR injury in the context of a complex natural Ab repertoire. Humans were also found to exhibit IgM natural Abs that recognize annexin IV. These data in toto identify annexin IV as a key ischemia-related target Ag that is recognized by natural Abs in a pathologic process required in vivo to develop intestinal IR injury.

Quantification of the mitochondrial DNA common deletion in presbycusis.

OBJECTIVES/HYPOTHESIS: This study was conducted to evaluate the association between the mitochondrial DNA (mtDNA) common deletion (CD) level in cochlear tissue and the severity of hearing loss in individuals with presbycusis. STUDY DESIGN: Nineteen individuals with presbycusis, ranging from 60 to 87 years of age, who met strict audiometric criteria were compared with four age frequency-matched normal hearing controls ranging from 51 to 76 years of age. Five additional normal hearing individuals, ranging from 9 to 50 years of age, were also studied. METHODS: A duplex real time polymerase chain reaction assay was used to quantify the mtDNA in archival cochlear tissue samples. Linear regression models were used for comparison of the CD level between groups. RESULTS: The presbycusis group had a mean CD level of 32% with a standard deviation of 14%, and the normal hearing age matched control group had a mean CD level of 12% with a standard deviation of 2%. This difference in CD levels reached statistical significance (P = .011) and remained significant after adjusting for any differences in age between the two groups (age-adjusted P = .007). Furthermore, there was evidence for a significant association between the CD level and the severity of hearing loss based on audiometric thresholds at 8 kHz (r = 0.44, P = .034; age-adjusted partial correlation = 0.55, P = .007). CONCLUSIONS: For the first time, to our knowledge, these results demonstrate a relationship between quantitatively measured levels of the CD in human cochlear tissue and the severity of hearing loss in individuals with presbycusis. Laryngoscope, 2009.

Technical report: laser microdissection of cochlear structures from celloidin embedded human temporal bone tissues and detection of the mitochondrial DNA common deletion using real time PCR.

Laser microdissection (LMD) has been used to isolate groups of cells and single cells from numerous tissues. In this study, we describe a technique for isolating cochlear structures and individual spiral ganglion cells from archival celloidin embedded human temporal bone sections. The specimens isolated are suitable for quantifying the mitochondrial DNA (mtDNA) common deletion (CD) within these tissues using a real time polymerase chain reaction (PCR) assay. The results presented in this manuscript demonstrate the feasibility of using this LMD technique to study the accumulation of mtDNA deletions in diseases of the ear. To our knowledge, this approach to analyzing archival human temporal bone tissues has not been previously reported.

Technical report: immunofluorescence and TUNEL staining of celloidin embedded human temporal bone tissues.

The large archival human temporal bone collections of the world have been fixed in formalin and embedded in celloidin. These treatments have created challenges to the use of contemporary probes, which are routinely used in the evaluation of fresh and frozen tissues, for the analysis of archival temporal bone tissues. Formalin alters the configuration of proteins and can obscure antigens by modifying the epitopes recognized by antibodies. Celloidin embedding provides superior support of the delicate membranous structures of the inner ear to maintain tissue integrity during sectioning, however, inadequate removal of celloidin may limit tissue permeability resulting in poor penetration of large molecules. Methods are described in this manuscript that have allowed reproducible immunofluorescence and TUNEL (terminal deoxynucleotidyl transferase mediated dUTP nick end labeling) staining results in these archival tissues. To our knowledge, successful immunofluorescence staining of type I collagen, immunofluorescence staining of cytochrome c oxidase subunit III (COX III), and TUNEL staining in archival human temporal bone tissues with confocal microscopy has not been previously reported. These results demonstrate the utility of developing techniques to evaluate the existing collections of archival temporal bones which remain our greatest source of tissue for investigating the causes of ear diseases.

Nasal ocular reflexes and eye symptoms in patients with allergic rhinitis.

BACKGROUND: Allergic patients often complain of eye symptoms during the allergy season. A possible mechanism for these eye symptoms is a nasal ocular reflex. OBJECTIVE: To demonstrate eye symptoms after nasal allergen challenge. METHODS: In a double-blind, placebo-controlled, crossover, clinical trial, 20 patients with seasonal allergic rhinitis were challenged in 1 nostril with antigen, and the response was monitored in both nostrils and in both eyes. Symptoms were recorded. Filter paper disks (intranasally) and Schirmer strips (intraocularly) were used for collecting secretions, which were subsequently eluted for the measurement of histamine and albumin levels. Patients were treated once topically at the site of challenge with azelastine or placebo. RESULTS: After placebo treatment, ipsilateral nasal challenge caused nasal symptoms and an increase in secretion weights; both were blocked by treatment with azelastine. Histamine and albumin levels increased only at the site of nasal challenge. Azelastine pretreatment inhibited the increase in albumin but not histamine levels. Symptoms of itchy and watery eyes increased significantly compared with symptoms with sham challenge after nasal allergen and were blocked by azelastine use. Ocular secretion weights increased bilaterally after placebo use and were not inhibited by azelastine use. CONCLUSIONS: Nasal allergen challenge releases histamine at the site of the challenge, which probably initiates a nasonasal and a nasal ocular reflex. This reflex is reduced by an H1-receptor antagonist applied at the site of the challenge. The eye symptoms associated with allergic rhinitis probably arise, in part, from a naso-ocular reflex.

Effect of topical ciprofloxacin/dexamethasone or dexamethasone alone on acute Streptococcus pneumoniae rhinosinusitis in mice.

OBJECTIVE: To test whether intranasal ciprofloxacin/dexamethasone or dexamethasone alone affects the course of acute bacterial rhinosinusitis in mice. STUDY DESIGN: We performed a randomized, double-blind, parallel, placebo-controlled study in mice. SUBJECTS AND METHODS: Three groups of 10 C57Bl/6 mice were infected with Streptococcus pneumoniae, and then 1 day later randomized to treatment with placebo, ciprofloxacin plus dexamethasone, or dexamethasone. The mice were killed 3 or 10 days after treatment was begun. RESULTS: The placebo-treated mice became infected and developed an inflammatory cell infiltration in their sinuses. None of the treatments significantly affected the course of the illness. CONCLUSION: The lack of topical, intranasal efficacy of ciprofloxacin and dexamethasone could be attributed to subpotent dosage, rapid nasal clearance, or inability of the drops to reach the site of infection. Treatment with dexamethasone neither improved nor worsened the bacterial infection.

Detection of mitochondrial DNA deletions in the cochlea and its structural elements from archival human temporal bone tissue.

Large-scale deletions of mitochondrial DNA (mtDNA) have been associated with aging and disease in post-mitotic tissues. These post-mitotic tissues, including skeletal muscle, heart and brain, are heavily dependent on intact functional mitochondria. The cochlear tissues are known to contain an abundance of mitochondria. This observation stimulated a search for mtDNA deletions in the cochlea and its elements using a sensitive nested PCR methodology and long range PCR to explain the functional deficits observed in age-related hearing loss. The presence of the so-called "common" deletion (CD) was detected in cochlear tissue from two individuals with age-related hearing loss, 73 and 78 years of age. Three additional deletions, that to our knowledge have not been previously reported, were also identified in these two individuals, including a 5354 bp deletion flanked with a 3 bp repeat, a 9682 bp deletion flanked by a 10 bp repeat and a 5142 bp deletion without a flanking repeat. The 9682 and 5142 bp deletions were also detected in an individual 39 years of age with normal hearing, however, these two deletions were not detected in a normal hearing individual 9 years of age. In contrast, the 5354 bp deletion was detected in all four of the individuals studied. To localize the deletions within the cochlea, the cochlear elements were removed by laser capture microdissection (LCM) and the mtDNA from these tissues was studied. The 5142 and 5354 bp deletions were detected in the organ of corti, spiral ligament, and ganglion cells, but not in the stria vascularis. These findings correlate with the reduction in the number of spiral ganglion cells and outer hair cells, and the normal stria vascularis volume observed in this individual. All four of these deletions involve the cytochrome c oxidase (COX) subunit III gene, encoded by mtDNA. These observations suggest that multiple mtDNA deletions may contribute to a deficit in mitochondrial function in the cochlea and result in hearing loss if a level of physiological significance is reached.

Distinct and separable roles of the complement system in factor H-deficient bone marrow chimeric mice with immune complex disease.

Plasma complement factor H (Cfh) is a potent complement regulator, whereas Cfh on the surface of rodent platelets is responsible for immune complex processing. For dissection between the two, bone marrow chimeras between Cfh-deficient (Cfh(-/-)) and wild-type C57BL/6 mice were created. Platelet Cfh protein was tracked with the Cfh status of the bone marrow donor, indicating that platelet Cfh is of intrinsic origin. In an active model of immune complex disease, Cfh(-/-) mice that were reconstituted with wild-type bone marrow had levels of platelet-associated immune complexes comparable to those of wild-type mice and were protected against the excessive glomerular deposition of immune complexes seen in Cfh(-/-) mice, yet these mice still developed glomerular inflammation. In contrast, wild-type mice with Cfh(-/-) bone marrow had reduced platelet-associated immune complexes and extensive glomerular deposition of complement-activating immune complexes, but they did not develop glomerular pathology. The large quantities of glomerular C3 in wild-type mice with Cfh(-/-) bone marrow were in the form of iC3b and C3dg, whereas active C3b remained in Cfh(-/-) recipients of wild-type bone marrow. These data show that plasma Cfh limits complement activation in the circulation and other accessible sites such as the glomerulus, whereas platelet Cfh is responsible for immune complex processing.

Evidence for binding of the ectodomain of amyloid precursor protein 695 and activated high molecular weight kininogen.

To identify ligands that bind to the N-terminal portion of human amyloid precursor protein (APP), we sought binding partners for a fragment of the ectodomain of human APP695 (sAPP(695)T). The probe bound to fragments of high molecular weight kininogen (HK) in rat cortical membrane preparations in vitro. Laser confocal microscopy indicated that APP and HK colocalize near cerebral blood vessels, in the neuropil, and in many neurons of rat brain. sAPP(695)T bound to human activated kininogen (HKa) (K(d)=0.3+/-0.1 nM), but not to inactivated or low molecular weight kininogen. Binding was specific for the light chain sequence of HKa. Biotinylated human HKa also bound to sAPP(695) (K(d)=0.3+/-0.5 nM). sAPP(695) and HKa form tight complexes in solution that can be coimmunoprecipitated. These results support the hypothesis that forms of APP and kininogen can interact in brain tissue. Considering the implications of APP in neurite outgrowth, the APP-HKa interaction could modulate neurogenesis.